The process of blood clotting begins with injury to a blood vessel. The damaged vessel wall initiates hemostasis by causing adherence and accumulation of platelets at the point of a vascular injury and by activating the plasma proteins which initiate the coagulation process. Sequential activation via specific proteolytic cleavages and conformational changes of a series of proteins (including Factor VIII) that comprise the coagulation cascade eventually leads to deposition of insoluble fibrin which, together with aggregated platelets, curtails the escape of blood through the point of injury in the damaged vessel wall. Specifically, Factor VIII is activated by thrombin which effects a proteolytic modification (cleavage) on the structure of Factor VIII (Weiss, H.J., et al, Science, 182:1149-51, 1973; Hoyer, L., Ch. 4, Hemostasis and Thrombosis, 2d Ed., Colman, R.W., Ed., J. B. Lipincott & Co., Philadelphia, USA, 1987; Fulcher et al, J. Clin. Invest., 6:117-24, 1985; and Eaton, et al, Biochemistry, 25:505-512, 1986).
Following the initial increase in activity after exposure of Factor VIII to thrombin, there is a decay in activity to below baseline levels. This decay in activity- is attributed to further proteolytic cleavage of activated FVIII by thrombin; (and during pathological states) inactivation by thrombin-activated protein C. The activation and inactivation of Factor VIII by thrombin and protein C allows a fine measure of control over coagulant activity in vivo. Unfortunately, it may also cause premature denaturation of Factor VIII prior to administration to mammalian hosts in need of such treatment.
The primary source of native Factor VIII is plasma. In addition, recombinant Factor VIII has recently become available through the cloning of the Factor VIII gene (Gitschier, J., et al, Nature, 312:326-330, 1984; Wood, W.I., et al, Nature, 312:330-337, 1984; and Vehar, G.A., et al, Nature, 312:337-342, 1984 and Toole, J.J., et al, Nature 312:342, 1984).
Recently, several groups of investigators have purified Factor VIII with specific activities as high as 3000 units per milligram (Rotblatt, F., et al, Thromb. Haemost., 50:108, 1983; Fulcher, C.A., et al, J. Clin. Invest., 76:117-124, 1985; Johnson, A.J., et al, Scripps Clinic and Research Foundation and the NHLBI, 1982).
The Factor VIII in all of the concentrates available to date (regardless of their purity) has an in vivo half-life of about 8-12 hours (Hoyer, L.W., Blood, 58:1-13, 1981) necessitating multiple infusions of concentrate for the maintenance of hemostasis. The level of Factor VIII antigen in plasma falls off at the same rate as the clotting activity, which suggests that Factor VIII is rapidly removed from the circulation and that the decline in Factor VIII activity is not simply or even primarily due to its inactivation.
The lower curve in FIG. 1 (which is a plot of the log of Factor VIII activity vs. time) shows a typical pattern of Factor VIII elimination from the bloodstream of a hemophilic dog. This pattern is similar to the one occurring in man. Phase 1 of the disappearance time curve is shown by the sharp nonlinear decrease in FVIII activity (to about 55% of the initial value) which follows its administration in vivo. This has been attributed to diffusion into the extravascular space or a more rapid removal of the higher molecular weight forms (Over et al., J. Clin. Invest. 62:223-234, 1978). Phase 2 of the disappearance time curve in FIG. 1, from which the half-life is determined, is demonstrated by the subsequent substantially linear decrease in activity. This second gradual decrease in activity has been attributed to clearance by the reticuloendothelial system and possibly protein C inactivation.
In this application, the term "disappearance time" or "clearance time" will be used to denote the time taken for FVIII activity to decrease to 50% of its maximum level. The maximum occurs in Phase 1 and the decrease to 50% may occur in Phase 1 or Phase 2.
Another problem incurred with infusion of Factor VIII stems from the fact that many recipients develop inhibitors (antibodies) which neutralize the activity of the infused Factor VIII. As many as 14% of the patients receiving Factor VIII infusions develop such inhibitors (Strauss, H.S., N. Engl. J. Med. 281:866, 1969). The presence and activity of these inhibitors are sometimes sufficient to eliminate the beneficial effect of Factor VIII infusion.
Attempts to bypass the effects of these inhibitors which include infusion of inhibitor-bypassing agents (e.g., Feiba made by Immuno, Austria and concentrates of Factors II, VII, IX and X) have been only partially successful. The bypassing agents themselves cause undesirable side effects and their mechanism of action is unknown. Immunosuppressive techniques have also been only partially successful usually in patients who have spontaneous anti-FVIII antibodies. However, administration of immunosuppressants raises the risk that the patient will b vulnerable to AIDS and opportunistic infections.
It would therefore be desirable to modify Factor VIII prior to i.v. infusion and thus: (a) prevent or lessen the initial, sharp decrease in Factor VIII activity in Phase 1 (ref. FIG. 1, lower curve) following the infusion of Factor VIII; (b) slow down the rate of the subsequent gradual decrease in Factor VIII activity in Phase 2 (and thereby prolong the Factor VIII half-life); (c) protect the infused Factor VIII from antibody inhibition by decreasing the ability of Factor VIII to bind to existing antibodies; (d) decrease the ability of the infused Factor VIII to generate further antibodies; and (e) modify the Factor VIII under conditions that would not substantially affect its overall sustained ability to participate in the coagulation cascade. The present invention meets one or more of these objectives in whole or in part as further described below.